Dopaminergic (DA) neurons control a variety of distinct brain functions and their malfunction or loss results in specific disease states. DA neurons are defined by the expression of a battery of specific terminal differentiation markers, including dopamine synthesizing enzymes and transporters. Little is known about how the expression of these terminal differentiation markers, and hence dopaminergic fate, is regulated. Understanding the regulatory mechanisms of DA neuron differentiation has wide-spread implications not only for basic but also clinical research. We propose here to employ the genetic amenability of the model system C. elegans, combined with state-of-the art technological advances, to genetically dissect the regulatory logic of DA neuron specification on a single neuron level in live animals through a combination of transgenic and genetic loss-of-function approaches. Our preliminary data has revealed the requirement of transcription factor (an ETS domain factor) and its cognate cis-regulatory target sequence, present in terminal markers of DA fate, for appropriate DA neuron differentiation. However, these regulatory components are not sufficient to explain the adoption of dopaminergic fate. We have obtained preliminary evidence for the involvement of other transcriptional regulators of the homeobox gene family and their cognate cis-regulatory motifs in controlling DA neuron differentiation in conjunction with the ETS domain factor and we test the hypothetical involvement of these homeobox genes by standard mutant analysis (Aim #1). Furthermore we use unbiased genetic mutant screens, which have already revealed several, as yet uncloned regulators of DA fate ("dopy" genes), to identify DA fate regulators in an unbiased manner (Aim #2). Factors found through our genetic approaches to be required for the regulation of DA fate will also be tested for whether they are sufficient to reprogram cells into a DA neuron-like state (Aim #3). PUBLIC HEALTH RELEVANCE: The project proposes to study the molecular mechanisms that control the development of dopaminergic neurons, a clinically important class of neurons. We use genetic approaches in the simple model organism C.elegans to address how the expression of genes that are normally expressed in dopaminergic neurons is regulated.